Study On The Lipid Synthesis And Accumulation Regulated By DGAT1 Gene During Pollen Development In Arabidopsis Thaliana

Triacylglycerol(TAG)is the main stored form of plant lipid,which is important for plant growth and development.The synthesis and metabolism of TAG are also very important for pollen development.Diacylglycerol transacylase(DGAT)encoded by DGAT1 gene of Arabidopsis thaliana is the only rate-limiting enzyme to catalyze and synthesize TAG.DGAT1 mutant as11 is researched and found that in seeds DGAT activity and TAG content decreased obviously,as well as fatty acid composition changed.Previous studies in our laboratory showed that the accumulation and distribution of polysaccharide and lipid during pollen development of mutant as11 was significantly different from WT.However,the regulatory characteristics and functions of DGAT1 gene on lipid accumulation in pollen development have not been reported in detail.In order to further explore and reveal the function of DGAT1 gene in lipid accumulation during pollen development,our research used semi-thin section and ultra-thin section technique to study the accumulation and distribution characteristics of polysaccharide and lipid during pollen development of as11 mutant,as well as the cytological characteristics of organelles related to lipid synthesis.And we used methanol/chloroform extraction,TLC and GC-MS to study the accumulation of total lipid and TAG in mature pollen of mutant as11,and the changes of fatty acid composition and content during pollen development.Real-time fluorescence quantitative PCR was used to study the changes in the expressions of genes related to the anabolism of sugars and lipid in the pollen development of WT and mutant as11.We aimed to analyze and discuss the regulation mechanism of DGAT1 gene on lipid metabolism in Arabidopsis thaliana during pollen development from cytology and molecular level.The main results are summarized as follows.1.During pollen development of WT and mutant as11,there are obvious black lipid particles appeared in tapeta of WT at vacuolated microspores stage.At the stage of binuclear pollen grains ?,both WT and mutant as11 accumulate lipid in pollen grains and tapeta.There are lipid drops distributed surrounding generative nuclei and starch grains deposited around vegetative nucleus in pollen grains.But content of lipid drops in as11 is less,while starch grains are more than those in WT.Amylopectin becomes the most stored starch type in as11.With pollen development,both WT and as11 continue to accumulate lipid drops inside pollen until pollens reach maturity.Mature pollens of WT contain abundant lipid drops,while lipid accumulation in as11 mature pollens is much lower than that in WT.2.During pollen development,the content of two organelles-elaiosome and lipidosome containing TAG,which is related to lipid synthesis appear later in as11 than in WT through ultrastructure results.And the content of endoplasmic reticulum which is connect with lipidosome synthesis in as11 is less than WT,leading to the content of lipidosome is lower in as11.This may reduce the materials for pollen to synthesize lipid in mutant,and lead to that the content of lipid drops in as11 is less than in WT eventually.3.In 100 mg freeze-dried mature anthers,the total lipid content of WT is 25.71 mg and TAG content of WT is 169.60?g.While the total lipid content of as11 is 18.88 mg and TAG content of as11 is 65.32?g,decreased by 26.57% and 61.48% than WT,respectively.Fatty acid composition and content of WT and as11 mature pollens have no obvious changes,and palmitic acid(C16:0)and linolenic acid(C18:3)are the main fatty acids.But the content of linolenic acid(C18:3)is much higher in as11 than in WT at Second mitosis stage.DGAT1 gene influenced TAG synthesis during pollen development and decreased the content of TAG and total lipid in mature pollens.Besides,it also increased the synthesis rate of linolenic acid and made its content reach the maximum at the stage of second mitosis in advance.4.DGAT1 directly affects the expression of genes related to starch and lipid synthesis during pollen development.At binuclear pollen grains ?,the expressions of PDCT,GPAT1 and GPAT6 are higher than in WT.At the second mitosis stage,only PAH2 are expressed at high levels in as11.However,there are no obvious differences in the expressions of other key genes except for GDPHc1,which was related to the transformation of sugars and lipids,is higher than that of WT at the mature pollen grains stage.The expression of genes in fatty acid synthesis was studied and found that,KAS? and FAB1 are expressed higher in as11 than in WT at vacuolated microspores stage.The expressions of KAS? and FAD3 synthesizing linolenic acid(C18:3)are higher in WT,however FAD2 synthesizing linoleic acid(C18:2)is expressed significantly higher than WT at this stage.Among starch synthase genes,especially SS gene family which is responsible for regulating amylopectin synthesis,SS1 and SS2 are expressed highly in as11 than WT at binuclear pollen grains ?.At mature pollen stage,the expressions of SS1,SS2 and SS3 are higher in as11,indicating that amylopectin as the main polysaccharide particles stored in as11 mature pollens.